This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. One of the major protein degradation pathways found in eukaryotic cells is the ubiquitin-mediated proteolysis system. The biological function of this system hinges critically on a class of enzymes designated the E3 ubiquitin ligases, which are responsible for the recognition of the protein substrates that are targeted for degrada-tion. Most E3 ligases are multi-subunit complexes that are assembled in a modular fashion. A family of proteins designated cullins function as scaffolds on which these complexes are assembled. Cullins are clearly required for the assembly of E3 complexes , but the manner in which these interactions are regulated is not understood. Cullins have been shown to be involved in a large variety of biological processes, including cell cycle control, removal of N-glycosylation containing proteins, transcriptional control, hormonal regulation, differentiation, development, and neurological disorders. This project is focused on one particular cullin, cullin 3 (Cul3), that has been shown to be involved in the degradation of cyclin E. Increased expression of cyclin E has been linked with the development of breast cancers. In a general sense it is becoming increasingly clear that the development of many cancers is profoundly influenced by abnormal proteolytic processes. Previous work has reconstituted in vitro ubiquitination reactions, and examined in vivo assembly of complexes. However, in vitro reactions have failed to recapitulate in vivo substrate specificity, and many important components of the E3 ligases remain to be comprehensively described, such as the substrate-selectivity subunits, or the crucial E2 ubiquitin conjugating enzymes. The proposed work is intended to address these important gaps in our knowledge through the identification of Cul3/E3 ligase complex components, the discovery of physiological substrates, and the analysis of the physical interactions between substrates and the Cul3/E3 complex. This work is expected to make a significant contribution towards our understanding of regulated proteolysis. Detailed progress reports are presented in the "Research Highlights" section.